1. Field of the Invention
The present invention relates to methods of producing gangliosides, e.g., GM1, from cells grown in culture. In particular, cells are treated chemically and/or biochemically manipulated to induce the production of gangliosides, e.g., GM1, and/or cells are cultured long-term at high density, without passaging, to accumulate gangliosides, e.g., GM1.
2. Background Art
GM1 Ganglioside Structure and Function
GM1 is a monosialoganglioside having the following structure:

GM1 is a constituent of nerve cell membranes, is known to modulate a number of cell surface and receptor activities, and plays important roles in neuronal differentiation and development, protein phosphorylation and synaptic function. GM1 therefore impacts neuronal plasticity and repair mechanisms, and the release of neurotrophins in the brain. In addition to its role in the nervous system, GM 1 is involved in internalization of pathogens, cell signaling, proliferation, survival and differentiation. It is a component of lipid rafts, a microdomain within the plasma membrane that is enriched in cholesterol and sphingolipids. Furthermore, GM1 is involved in activation of a sodium-calcium exchanger in the inner membrane of the nuclear envelope. Its interaction with the calcium exchanger modulates nuclear and cellular calcium. In addition to its function in cellular physiology, GM 1 acts as the site of binding for cholera toxin.
GM1 has been shown to be effective in treating different types of central nervous system lesions in experimental animals, resulting in significant biochemical and behavioral recovery. Moreover, pretreatment with GM1 inhibits damage resulting from a variety of neurotoxin exposures.
GM1 has also been shown to be effective in the short-term treatment of Parkinson's disease subjects, resulting in significant symptom reduction. Schneider et al., Neurology 50:1630-1636 (1998). A more recent five-year study indicates that long-term GM 1 use by Parkinson's disease subjects is safe and may provide some clinical benefit for these subjects. Schneider et al., J. Neurol. Sci. 292:45-51 (2010), incorporated herein by reference in its entirety. It is uncertain how GM1 exerts potential neuroprotective, neurorestorative or neurorescue effects on the dopamine system. Id. at 50. However, it is speculated that GM1 incorporated in neuronal plasma membranes may alter the stability of lipid rafts and therefore promote a variety of beneficial cellular processes. Id.
Gangliosides
Gangliosides are a major glycosphingolipid in mammals, containing sugar chains with different numbers of sialic acid residues. Many different subspecies of sugar exists in gangliosides. Gangliosides are implicated in a number of diseases and disorders, including Tay-Sachs disease, Parkinson's disease, Alzheimer's disease and cancer, among others.
The biosynthesis of gangliosides are closely interconnected through the use of common biosynthetic enzymes and substrates. For example, the production of GM1 relies on the enzyme galactosyltransferase II, commonly used to produce other gangliosides, e.g., GA1, GD1b and GT1c. Xu et al., J. Lipid Res. 51:1643-1675 (2010), incorporated herein by reference in its entirety. Because of their common structural features and components, new gangliosides are often synthesized from recycled components of degraded gangliosides, in particular ceramide and sphingosine. Id. For example, core molecules such as ceramide, galactose, GalNAc, sialic acid, are required for synthesis of gangliosides. Id. As a result, factors that influence the production or degradation of one member of the ganglioside family frequently alter the production and degradation of other gangliosides. For example, because GM1 is the precursor to GD1a, increases in GM1 will favor the production of GD1a for the cell to maintain a normal or balanced proportion of gangliosides. Mason et al., Biochem. J. 388:537-544 (2005); Miller-Podraza et al., Biochem. 21:3260-3265 (1982); Nishio et al., J. Biol. Chem. 279:33368-33378 (2004), each of which is herein incorporated by reference in its entirety.
GM1 Production
GM1 derived from the bovine brain has been used clinically in the past. See, e.g., Schneider et al., J. Neurol. Sci. 292:45-51, 46 (2010) (“Patients self-administered . . . bovine brain-derived [GM1] sodium salt . . . ”), incorporated herein by reference in its entirety. However, the limited yield of GM1 per bovine brain and the cost of producing GM1 in this manner has restricted the amount of GM1 available for commercial clinical use. In addition, diseases such as bovine spongiform encephalopathy, i.e., mad cow disease, have raised concerns regarding the safety of this source of GM1. While extraction of GM1 from the brains of sheep afflicted with GM1 gangliosidosis has also been described (see, e.g., U.S. Pat. No. 5,532,141), incorporated herein by reference in its entirety, such a method raises similar concerns regarding yield, cost and safety.
A clear, unmet need therefore exists for a cost-effective, high-yield and safe alternative to making GM1 for commercial clinical use.